Our long term goal is to identify and characterize the genes involved in the pathogenesis of keratoconus. Keratoconus is the most common corneal ectatic disorder with onset at puberty. It is characterized by progressive corneal thinning and protrusion of the thinned corneal, which leads to high myopia and irregular astigmatism. It is listed as a significant public health concern and a research priority by the National Institute of Health - National Eye Institute. Family and twin studies including linkage analyses indicate the genetic contribution to the pathogenesis of keratoconus. Mutations in the VSX1 gene and miR-184 have been identified, but only account for a small number of patients. The majority of genetic mutations in keratoconus remain to be identified. We hypothesize that mutations in coding regions account for most of the familial patients. In our preliminary studies, we already collected 22 multiplex families from Saudi Arabia with a high degree of consanguinity. All the patients were screened for mutations in the two known genes. We have performed whole exome sequencing in four patients from two families, filtered and prioritized variants to less than 50 candidates in both families. In Aim 1, we will continue to perform whole exome sequencing in other families. We will perform homozygosity mapping using Illumina BeadChips, a powerful approach to mutation discovery in inbred populations. In addition to the families from Saudi Arabia, we will study multiplex families newly enrolled at Duke University Eye Center. We expect to collect 5-6 multiplex families and 40-50 sporadic patients per year through Duke Eye Center, from over 1,200 already diagnosed patients locally. We have a free monthly event Keratoconus Connection: Education Peer Group Session with patients, which will further ensure the success of our clinical ascertainment. In Aim 2, we will identify genetic mutations due to DNA copy number changes (i.e. genomic deletion or duplications) in these families. Our rationale is based on two lines of evidence. First deletions and duplications in a linkage locus- chr5q31 have been associated with keratoconus. Second, significantly increased prevalence of keratoconus in Down syndrome patients (three copies of chromosome 21) also indicates the role of DNA duplication. We will analyze data from Illumina BeadChips in Aim1 to examine DNA deletions and duplications. Family segregation as well as gene expression in human cornea will be used to prioritize the candidate DNA deletions and duplications. We will use Realtime PCR to validate the selected candidates. Mutations identified in Aim1 and Aim2 will be validated and replicated in a combined dataset with 140 multiplex families, 980 sporadic cases and 3700 examined controls. In summary, upon successful completion, we will identify genetic mutations in familial keratoconus, thus significantly improving our understanding of the disease and providing novel targets for diagnosis and treatment.